Evaluation of newly synthesized 2-(thiophen-2-yl)-1H-indole derivatives as anticancer agents against HCT-116 cell proliferation via cell cycle arrest and down regulation of miR-25

In the present study, we prepared new sixteen different derivatives. The first series were prepared (methylene)bis(2-(thiophen-2-yl)-1H-indole) derivatives which have (indole and thiophene rings) by excellent yield from the reaction (2 mmol) 2-(thiophen-2-yl)-1H-indole and (1 mmol) from aldehyde. The second series were synthesized (2-(thiophen-2-yl)-1H-indol-3-yl) methyl) aniline derivatives at a relatively low yield from multicomponent reaction of three components 2-(thiophen-2-yl)-1H-indole, N-methylaniline and desired aldehydes. The anticancer effect of the newly synthesized derivatives was determined against different cancers, colon, lung, breast and skin. The counter screening was done against normal Epithelial cells (RPE-1). The effect on cell cycle and mechanisms underlying of the antitumor effect were also studied. All new compounds were initially tested at a single dose of 100 μg/ml against this panel of 5 human tumor cell lines indicated that the compounds under investigation exhibit selective cytotoxicity against HCT-116 cell line and compounds (4g, 4a, 4c) showed potent anticancer activity against HCT-116 cell line with the inhibitory concentration IC50 values were, 7.1±0.07, 10.5± 0.07 and 11.9± 0.05 μΜ/ml respectively. Also, the active derivatives caused cell cycle arrest at the S and G2/M phase with significant(p < 0.0001) increase in the expression levels of tumor suppressors miR-30C, and miR-107 and a tremendous decrease in oncogenic miR-25, IL-6 and C-Myc levels. It is to conclude that the anticancer activity could be through direct interaction with tumor cell DNA like S-phase-dependent chemotherapy drugs. Which can interact with DNA or block DNA synthesis such as doxorubicin, cisplatin, or 5-fluorouracil and which were highly effective in killing the cancer cells. This data ensures the efficiency of the 3 analogues on inducing cell cycle arrest and preventing cancer cell growth. The altered expressions explained the molecular mechanisms through which the newly synthesized analogues exert their anticancer action.

We optimized the reaction conditions in various solvents, temperatures, and catalysts (Table 1) using benzaldehyde (3a) as the model substrate.(Fig. 2) When we used PPA/SiO 2 (0.11) or HClO 4 /SiO 2 (0.11) as catalyst in water under reflux for 10 min the reaction completion detected by TLC the yield of compound 4a and 5a approximately 60 and 15%, respectively.On the other hand, when the reaction is occurred by using SSA (0.11) as a catalyst without solvent the reaction completed after 30 min and the yield is relatively low.The reaction occurred by using SSA (0.11) in different solvents under reflux.The best yield is formed by using water as solvent and the reaction completed in the least time.When we decrease the concentration of SSA and fixed the solvent and the temperature the yield decreases.So, the best result was obtained by using SSA (0.11 mmol) under reflux in water the reaction completed after 5 min.

Evaluation of in vitro cytotoxic activity of analogues
The screening of newly synthesized derivatives on different cancer monolayer cell lines is illustrated in Table 3.
The initial screening of newly synthesized derivatives were performed at a single dose concentration of 100 ppm against a panel of 5 different cancer cell lines namely, human breast cancer (MCF-7), human colon cancer (HCT-116 and HT-29), human skin cancer (A375) and Human non-small cell Lung cancer (A549).Test compounds were administered to the cell lines and after 48h the percent cytotoxicity of treated cells was determined relative to untreated cells, Doxorubicin was used as positive control.Results of the initial single dose (100 µg/ ml) testing for all 15 compounds against this panel of 5 human tumor cell lines indicated that the compounds under investigation exhibited selective cytotoxicity against HCT-116 and HT-29 cell line as compared to the reference doxorubicin, data are illustrated in (Fig. 5).Surprisingly, the compounds showed no activity against other cell lines (data not shown).HCT-116 cell line was the most sensitive to our compounds.Results showed   3, Fig. 5).

Selectivity Index (SI)
Compounds that showed potent cytotoxic (≥ 60%) effect were subjected to dose response study to calculate the inhibitory concentration 50(IC 50 ).4g, 4a, 4c compounds appeared also to be both the most potent and most selective against HCT-116 cells with an SI > 2.74 (SI: 13, 9.7 and 2.4, respectively) (Table 3).This finding encouraged us to further study the cellular mechanisms underlying the potent cytotoxic effect observed against colorectal cancer cells.The observation that these compounds possess high selectivity against cancer cells and low toxicity to normal cells highlights their potential as potent and safe anticancer drugs for further studies.

Molecular docking study
To determine the possible mechanisms of the anticancer activity of the best 3 synthesized compounds (4g, 4a, 4c) a molecular-docking study was done.We investigated their binding affinity against c-Myc and IL-6.These proteins are vital targets to generate anticancer agents.They were selected due to their vital role in the carcinogenesis process, consequently, targeting these proteins will be a good strategy for inventing efficient anticancer agents.The binding free energy of ligands and selected target proteins, number and type of interactions are summarized in Table 4, Fig. 6 while Figs. 7, 8 showed the two and three dimensional interactions between the ligands and the selected proteins.It was observed that all docked ligands had RMSD values < 2, which ensures proper docking protocol.All three ligands showed high binding affinity with both proteins which ensures their potency as inhibitors for c-Myc and IL-6 proteins.However, 4g had the best binding free energy (− 10.5 and − 8.3 kcal/mol) with both proteins c-Myc and IL-6 respectively.Many types of interactions were observed such as Hydrogen bond, Pi-Anion, Pi-Pi T shaped, Pi-Alkyl, Alkyl, Van Der Waals which ensures the high binding affinity between synthesized compounds and target proteins.This diverse interaction profile contributes to the ligands' high binding affinity and strengthens their binding to the protein surfaces.Pro637, Glu701, Glu640, and Phe729 of c-Myc protein were involved in many interactions with all three ligands while Arg179, Ser176 were the most interacting in case of IL-6 protein.Docking results ensured the potency of 4g, 4a, 4c as anticancer agents especially in colorectal cancer and showed the possible mechanism through which these agents may exert their anticancer effect.www.nature.com/scientificreports/

Cell cycle analysis and quantitative RT-PCR
Numerous studies have been reported that some anti-cancer agents stimulated cell cycle arrest and thereby inducing apoptotic cell death.In particular, the uncontrolled cell cycle is a hallmark of tumor cells, and it is contributed to the progression and development of cancer [29][30][31][32] .Our results showed that treatment of HCT116 cells with 4g, 4c and 4a showed a significant increase in the percentage of cells in the S and G2/M phases compared to control (Fig. 9).These results indicated that our analogues induced cell cycle arrest at the S and G2/M phase.This would suggest that the compounds interfere with processes necessary for DNA replication or mitosis, effectively halting cell division.This is a potential anti-cancer mechanism, as uncontrolled cell division is a hallmark of cancer so, their anticancer activity could be through direct interaction with tumor cell DNA like S-phase-dependent chemotherapy drugs, which can interact with DNA or block DNA synthesis such as doxorubicin, cisplatin, or 5-fluorouracil and which were highly effective in killing the cancer cells [33][34][35] .Doxorubicin is known to induce cell cycle arrest in HCT116 cells, primarily at the G2/M checkpoint 36 .Also in other cell lines as MDA-MB-231 cells when treated with Dox resulted in a significantly higher percentage of cells in the G2M phase 37 .This arrest prevents cells from entering mitosis, thereby halting cell division and promoting cell death.Our results showed that treatment of HCT116 cells with 4g, 4c, and 4a caused a significant increase in the percentage of cells in the S and G2/M phases compared to the control (Fig. 9).These findings suggest that our analogues, like doxorubicin, might induce cell cycle arrest, potentially at the S and G2/M phases.This data highlights the efficiency of the 3 analogues on inducing cell cycle arrest and preventing cancer cell growth.
To further investigate the molecular mechanism through which our drugs exert their anticancer effect, a panel of miRNAs and genes which play a crucial role in colorectal cancer cell proliferation, tumorigenesis, and metastasis were selected: 3 miRNAs (miR-25, miR-30C, and miR-107) and two genes (IL-6 and C-Myc).It was observed a significant upregulation of miR-25, IL-6 and C-Myc (p < 0.001) while miR-30C, and miR-107 were downregulated (p < 0.001) in control HCT116 cells.The upregulation of miR-25, IL-6, and C-Myc in control HCT116 cells aligns with their oncogenic roles.Increased miR-25 promotes cell proliferation and inhibits apoptosis, while elevated IL-6 and C-Myc levels drive tumor growth and metastasis.Downregulation of tumor suppressors miR-30C and miR-107 further exacerbates the cancerous phenotype.The treatment of HCT116 cells with the 3 drugs caused a dramatic reversal by totally altering the expression pattern (p < 0.0001) as shown in     Our data are in accordance to many studies as the tumor suppressors miR-30C, and miR-107 are significantly downregulated in CRC both in cell lines and cancer tissues compared with matched normal adjacent tissue and this downregulation is related to shorter overall survival 38 .MiR-30C, and miR-107 are highly important for suppression of tumor metastasis, inhibition of cancer cell growth, migration and invasion [39][40][41][42] .In contrast, oncogenic miR-25 expression is significantly elevated in colorectal cancer and associated with tumor invasion, lymph node metastasis, distant metastasis and TNM.In addition, an increased level of miR-25 expression is associated with a poor overall survival of patients 43,44 .Regarding selected genes, the c-Myc gene is a nuclear transcription factor that mainly regulates cell growth, cell cycle, metabolism, and survival 45,46 which is frequently overexpressed in approximately 70% of human cancer and related to metastatic progression of colorectal cancer [47][48][49] .Several experimental and clinical studies have linked the pleiotropic cytokine interleukin-6 (IL-6) to the pathogenesis of sporadic and inflammation-associated colorectal cancer (CRC).Increased IL-6 expression has been related to advanced stage of disease and decreased survival in CRC patients 50,51 .IL-6 level was significantly higher in patients with colorectal cancer than in normal controls.High levels of serum IL-6 were correlated with larger tumor size, elevated serum CRP levels, and liver metastases.IL-6 levels also increased in a stage-related manner [52][53][54] .
Based on the previous results, 4g, 4c, and 4a exhibit potent and multi-pronged antitumor activity against HCT116 colorectal cancer cells.These promising agents directly interact with cellular DNA, causing cell cycle arrest at S and G2/M phases, effectively halting tumor cell proliferation.Furthermore, they upregulate tumor suppressor miRNAs miR-30C and miR-107, which inhibit cancer cell growth and metastasis.Concurrently, they downregulate oncogenic miR-25, c-Myc, and IL-6, known to promote tumorigenesis and shorten patient survival.This multifaceted approach, targeting both DNA and key regulatory molecules, suggests 4g, 4c, and 4a hold significant potential for colorectal cancer treatment.Further investigations, including in vivo studies and target gene identification, are crucial to validate these findings and pave the way for future therapeutic applications.

Chemistry
Melting points were determined with an electro thermal digital melting point apparatus (Electro-Thermal Engineering Ltd., Essex, United Kingdom).The IR spectra were recorded in KBr disks on a Pye Unicam SP 3300 and Shimadzu FT IR 8101 PC Infrared Spectrophotometers (Pye Unicam Ltd.Cambridge, England and Shimadzu, Tokyo, Japan, respectively).1H and 13C NMR spectra were obtained from a Jeol ECA 500 MHz NMR Spectrometer (Tokyo, Japan) using deuterated dimethyl sulfoxide (d6-DMSO) as a solvent and (TMS) as an internal reference at 500, 125 MHz, respectively spectra were obtained from a JEOL ECA 500 MHz NMR Spectrometer at 200 MHz Mass spectra (EI-MS) were obtained with ISQ (Single Quadrupole MS, Thermo Scientific).Elemental www.nature.com/scientificreports/analyses (C, H, N) results were recorded with Elementar Vario EL Germany.The recorded yields are of pure isolated materials obtained by column chromatography silica gel 60 (Merck) and thin layer chromatography (TLC) which was performed on Merck Kiesel gel F254 precoated plates (Merck, Darmstadt, Germany).

Cell culture
The human (HCT-116, HT-29, A549, MCF-7 and A375) cancer cell lines and RPE-1, normal cell line were maintained in DMEM-F12 medium.The media was supplemented with 10% fetal bovine serum at 37 °C in 5% CO 2 and 95% humidity.Cells were sub-cultured using trypsin versene 0.15%.The anticancer activity was conducted at the Bioassay-Cell Culture Laboratory, National Research Center, Cairo, Egypt, for in vitro primary antitumor screening on Human Colorectal carcinoma (HCT-116, HT-29) and Normal Human Retinal Epithelial (BJ-1) cell line.All cell lines were kindly provided by Professor Stig Linder, Oncology and Pathology department, Karolinska Institute, Stockholm, Sweden, originally obtained from ATCC.

Cytotoxicity on cancer monolayers
The cytotoxic assay on cancer cell lines was done according to the method of 55,56 with slight modifications.All the following procedures were performed in a sterile area using a laminar flow cabinet bio-safety class II level (Baker, SG403INT, and Sanford, ME, USA).Briefly, after 24 h of seeding 10,000 cells per well for HCT-116, HT-29, A549, MCF-7 and A375 cancer cell lines (in 96 well plates), the medium was changed to fresh medium and cells were treated with 100 μg/ml final concentration of the different compounds in triplicates for 48 h. 100 μM doxorubicin was used as positive control and 0.5% DMSO was used as negative control.Cell viability was determined using the MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay as described previously 57 .
The percent cytotoxicity was calculated according to the following equation: %cytotoxicity = [1 − (AV x /AV NC )] × 100 where AV: average, X: absorbance of sample well, NC: absorb- ance of negative control measured at 595 nm with reference at 690 nm.

Determination of IC 50 values
Compounds that showed highly active cytotoxicity on different tested cell lines were selected for dose response study at different concentrations.The final tested concentrations were 100, 50, 25, 12.5, 6.25 μg/ml and up to 0.78 μg/ml, in triplicates.The IC 50 values were calculated using the concentration-response curve fit to the nonlinear regression model using GraphPad Prism® v6.0 software (GraphPad Software Inc., San Diego, CA, USA).

Selectivity Index (SI)
The selectivity index (SI) indicates the cytotoxic selectivity (i.e., safety) of different compounds against HCT-116 cancer cells versus normal cells (RPE-1).SI = IC 50 of compound in the normal cell line/IC 50 of the same compound in the cancer cell line.The higher the SI value, the higher the safety of the compound.Compounds possessing SI value > 2 are considered 58,59 .

Molecular docking study
The structures of all synthesized compounds (ligands) were displayed by the ChemDraw JS sample online page.The ligands were prepared for docking by optimization, and energy minimization using the PyRx software 60 .The three-dimensional crystal structures of the target proteins were downloaded from Protein Data Bank (PDB): HUMAN INTERLEUKIN-6 (PDB ID: 1ALU), c-Myc (PDB ID: 5VHE).The target proteins were prepared for docking by removing water and ions, adding hydrogen, and the allocation of charge.The preparation process was achieved by UCSF Chimera version 1.11.2 61 .The active sites were distinguished with proper grid boxes round the bound crystallized ligands.The docking study was achieved by PyRx software 60 , where the less energy isomers were generated and the best ligand-receptor complexes were assessed according to binding affinity.The Discovery Studio Visualizer program was used for visualization of docked complexes 62 .

Cell cycle analysis by flow cytometry
Cell cycle analysis was carried out to detect the probable changes in the cell cycle phases in control and treated cells. 1 × 10 6 of HCT116 cells suspended in 0.5 ml 1 × DPBS and were aspirated several times with Pasteur pipet and fixed with 70% ethanol on ice for 2 h and centrifuged for 5 min at 300 × g.Cell pellets were re-suspended in 5 ml 1 × DPBS for 30 s and centrifuged at 300 × g for 5 min then re-suspended in 1 ml of PI staining solution and kept in the dark at room temperature for 30 min.Finally, cells were then transferred to the CytoFLEX Flow Cytometer (Beckman Coulter Life Sciences, USA) to measure the cell fluorescence (BECKMAN COULTER Inc., Cairo, Egypt and Cat.No. 4238055-CB) for cell cycle analysis.The percentage of cells in G0/G1, S, and G2/M phases of the cell cycle was calculated using CytExpert Software 63,64 .

RNA extraction and quantitative real time PCR
After treatment with 4g, 4a, and 4c for 48h, HCT116 cells were harvested and total RNAs including small sequence RNAs (miRNAs) were extracted from cell pellets using miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol.RNAs were quantified at A260/A280 nm using a NanoDrop1000 (Thermo Fisher Scientific GmbH, Dreieich, Germany).Extracted RNAs were reverse-transcribed, using miScript everse transcription Kit (Qiagen) for miRNAs and RevertAid Reverse Transcriptase kit (Thermo Scientific) for genes.The differential expression levels of the selected miRNAs and genes were analyzed by quantitative real-time PCR (qRT-PCR) using miScript SYBR green PCR kit (Qiagen) on a real-time PCR machine (Agilent, Mx3000P, CA, USA).RT-PCR was performed in triplicates using the following cycling conditions: 95 °C for 15 min, followed by 40 cycles of 94 °C for 15 s, 55 °C for 30 s, and 70 °C for 30 s.All samples were normalized to the internal control RNU6 for miRNAs and GAPDH for genes and relative expression levels were calculated with the 2−ΔΔCt method and represented as fold change.

Figure 5 .
Figure 5. Average % cytotoxicity of different compounds on HCT-116 colon cancer cell line.Cell viability was determined after 48h and evaluated by MTT assay.Doxorubicin (DOX.) was used as positive control.Values are expressed as mean ± SD, n = 3 at a concentration of 100 µg/ml.

Figure 6 .
Figure 6.Number of interactions of the ligands with c-Myc and IL-6 proteins.

Fig. 10 .
Fig.10.The treated cells showed a significant increase in the expression levels of tumor suppressors miR-30C, and miR-107 and a tremendous decrease in oncogenic miR-25, IL-6 and C-Myc levels.These results suggest that our drugs may act through multi-pronged mechanisms, simultaneously repressing oncogenes and activating tumor suppressors.This multifaceted approach could potentially overcome drug resistance and lead to more effective cancer treatment.

Figure 7 .
Figure 7. Two dimensional interactions of the best docked poses of the selected compounds with c-Myc and IL-6 proteins.

Figure 8 .
Figure 8. Three dimensional model of the best docked complexes inside the binding site of C-Myc and IL-6 proteins.

Figure 9 .
Figure 9. Histograms of cell cycle phase distribution by flow cytometry using PI staining and Relative DNA content for HCT-116 cells.(A) untreated HCT-116 cells; (B) HCT-116 cells treated with compound 4a; (C) HCT-116 cells treated with compound 4c; (D) HCT-116 cells treated with compound 4g.The HCT-116 was plated at density of 1 × 10 6 cells/well in 6-well plate.The cells were treated with IC 50 value for each compound and incubated at 37 ℃ for 48h.

Figure 10 .
Figure 10.The effect of 4g, 4c and 4a treatment on HCT116 cells by qRT-PCR.A) Relative expression of miR-25, miR-30C, and miR-107 in control and treated HCT116 cells.B) Relative expression of c-Myc and IL-6 in control and treated HCT116 cells.* means that p value < 0.05, ** means that p value < 0.01, *** means that p value < 0.001.

Table 2 .
The reaction time, m.p. and yield for compounds (

Table 3 .
Dose-response data of active compounds tested against HCT-116 cancer cell lines and the normal cell line RPE-1 after 48 h.IC50, Inhibitory concentration 50 in μg/ml; SI, selectivity index; NA, not active.Doxorubicin was used as positive control drug.

Table 4 .
Docking score and the best interactions for the ligand compounds with target proteins.